Expanding Orbitrap Collision Cross-Section Measurements to Native Protein Applications Through Kinetic Energy and Signal Decay Analysis

Abstract

The measurement of collision cross sections (CCS, σ) offers supplemental information about sizes and conformations of ions beyond mass analysis alone. We have previously shown that CCSs can be determined directly from the time-domain transient decay of ions in an Orbitrap mass analyzer as ions oscillate around the central electrode and collide with neutral gas, thus removing them from the ion packet. Herein, we develop the modified hard collision model, thus deviating from the prior FT-MS hard sphere model, to determine CCSs as a function of center-of-mass collision energy in the Orbitrap analyzer. With this model, we aim to increase the upper mass limit of CCS measurement for native-like proteins, characterized by low charge states and presumed to be in more compact conformations. We also combine CCS measurements with collision induced unfolding and tandem mass spectrometry experiments to monitor protein unfolding and disassembly of protein complexes and measure CCSs of ejected monomers from protein complexes.

Determining Collisional Cross Sections from Ion Decay with Individual Ion Mass Spectrometry

“…Isotopic resolution is readily achieved for all proteins besides alcohol dehydrogenase and pyruvate kinase in Figure and could be achieved for larger protein complexes by extending the acquisition time to increase the number of ions collected . The Ecm correction factor determined herein showed stability over the course of the study (∼8 months), which is consistent with the correction factor developed by James et al This approach may also be applied to previously collected data using DMTmode if sufficient ion decay was observed. Determining CCS by I 2 MS provides an orthogonal measurement that may be extracted from I 2 MS data sets without increasing the experimental time required, allowing for more detailed characterization of proteins and protein complexes with Orbitrap mass spectrometers.…”

“…Dissociation or dephasing events for denatured proteins typically occur after an ion collides with a single gas molecule, but native proteins may endure many more collisions. This results in underestimated CCS by eq , and the extracted CCS values for native proteins require additional correction by eq . After calculating raw CCS values for native proteins, the percent deviation between the Orbitrap CCS and IMS CCS values is plotted in Supplemental Figure S4 to determine the Ecm correction factor in eq .…”

“…Denatured CCS were determined using eq (described by Sanders et al), where c is the decay constant (1/s), L is the average path length of the ion during one oscillation (m), f z is the frequency of the ion (Hz), and n g , the density of gas molecules, was determined for denatured analytes by calibrating with the ubiquitin 9+ charge state as previously described and by the ideal gas law for native analytes. − The equations for converting from m / z to frequency for both instruments used are available in Supplemental Scheme 1. corrected CCS = uncorrected CCS 1 − ( Ecm * − 9.6 + 223.24 100 ) Native CCS were determined using the collision energy correction in eq as previously described by James et al The center of mass collision energy (Ecm) for a protein charge state is used to estimate the percent difference between the Orbitrap CCS measurements and IMS CCS measurements to correct values from eq . Native CCS were ultimately calculated by weighted average using the charge state CCS and charge state ion count as the literature shows that native protein CCS does not change significantly between charge states. ,− …”

“…Recently, methods have been developed for determining protein CCS directly from FT-MS data, including FT-ICR and Orbitrap systems, without additional IMS instrumentation. − These methods monitor dephasing events (i.e., in the form of signal decay) that remove ions from stable orbitals during mass analysis. Determining CCS from FT-MS signal provides a unique alternative to IMS systems and opens the path for integrating CCS methods with other modes of FT-MS analysis.…”

Investigating lipid transporter protein and lipid interactions using variable temperature electrospray ionization, ultraviolet photodissociation mass spectrometry, and collision cross section analysis

“…53,54 All CCS data were calibrated to myoglobin (8+ charge state, ion mobility CCS 1966 Å 2 ) collected at the same pressure and on the same day, and CCSs were fitted owing to extended ion survival as previously described. 55 Variable temperature ESI data was analyzed using a custom MATLAB R2020a script as previously described. 51 All CCS and variable temperature ESI data were collected in triplicate, and error bars represent the standard deviation of these replicates.…”